The structural analysis of shark IgNAR antibodies reveals evolutionary principles of immunoglobulins

Abstract

Sharks and other cartilaginous fish are the phylogenetically oldest living organisms that rely on antibodies as part of their adaptive immune system. They produce the immunoglobulin new antigen receptor (IgNAR), a homodimeric heavy chain-only antibody, as a major part of their humoral adaptive immune response. Here, we report the atomic resolution structure of the IgNAR constant domains and a structural model of this heavy chain-only antibody. We find that despite low sequence conservation, the basic Ig fold of modern antibodies is already present in the evolutionary ancient shark IgNAR domains, highlighting key structural determinants of the ubiquitous Ig fold. In contrast, structural differences between human and shark antibody domains explain the high stability of several IgNAR domains and allowed us to engineer human antibodies for increased stability and secretion efficiency. We identified two constant domains, C1 and C3, that act as dimerization modules within IgNAR. Together with the individual domain structures and small-angle X-ray scattering, this allowed us to develop a structural model of the complete IgNAR molecule. Its constant region exhibits an elongated shape with flexibility and a characteristic kink in the middle. Despite the lack of a canonical hinge region, the variable domains are spaced appropriately wide for binding to multiple antigens. Thus, the shark IgNAR domains already display the well-known Ig fold, but apart from that, this heavy chain-only antibody employs unique ways for dimerization and positioning of functional modules. protein evolution | antibody structure | protein folding | protein engineering

A current perspective on the compensatory effects of urea and methylamine on protein stability and function

“…Urea, although a waste product is also accumulated in the mammalian kidney at very high concentrations (up to 3-4 M) and contributes in osmo-adaptation (Yancey, 1988;Yancey, 1994;Yancey, 2001;Yancey et al, 1982). For the survival of these animals under such large concentration of urea, three adaptational hypothesis have been suggested which include the urea insensitiveness of some proteins; for instance, actomyosin ATPases of elasmobranchs and IgNAR (Ig new antigen receptor) antibodies of sharks are more stable in urea (Bonaventura et al, 1974;Feige et al, 2014;Hasnain and Yasui, 1986;Yancey, 1985); urea requirement by some proteins, for instance, in case of lactate dehydrogenase and eye lens protein of elasmobranches wherein urea was found to maintain optimum K m for pyruvate and prevented the precipitation of the latter at low temperatures (Yancey and Somero, 1978;Zigman et al, 1965); and urea-counteracting osmolyte system; for instance, the activity of an enzyme in the presence of urea is maintained by the co-accumulation of another stabilizing osmolyte (Yancey, 1988;Yancey et al, 1982;Yancey and Somero, 1979;Yancey and Somero, 1980). Among these three, the last one seems to be the primary one.…”

Green Turtles (Chelonia mydas) Have Novel Asymmetrical Antibodies

“…Although unconventional Abs have been seen in other animals such as H chain-only Ig in camels (5), or natural Ag receptors in sharks (34), all of these have been bilaterally symmetrical. Humans have asymmetric Igs with a different glycosylation pattern in one of the Fab arms, making them univalent and unable to precipitate Ags; these Abs play important roles in maternal immunity (35).…”

Technology advancements in antibody purification

“…Today, naturally occurring immunoglobulin of all shapes and sizes have been discovered in mammals (IgG, IgE, IgA, IgD, and IgM), and numerous other formats occur throughout the animal kingdom (eg, IgY from birds, single-domain antibodies from camelids, and heavy-chain-only antibodies from cartilaginous fish). 7 Apart from naturally occurring antibody formats, combinatorial deoxyribonucleic acid (DNA) technology has facilitated the development of recombinant antibodies such as fragment variable (Fv), disulfide-stabilized Fv antibody fragment, single-chain fragment variable (scFv), fragment antigenbinding (Fab), single-chain antibody fragment, divalent antibody formats such as minibody, diabody, F(ab′) 2 and (scFv) 2 , and multivalent forms such as tetrabodies, triomabs, triabodies, and F(ab) 3 . 8,9 Overview of conventional methods Selective antibody purification is commonly carried out using affinity chromatography or affinity-tag-based chromatography.…”